The human ether-a'-go-go related gene (hERG) K+ channel blockade by the investigative selective-serotonin reuptake inhibitor CONA-437: limited dependence on S6 aromatic residues.

Diverse non-cardiac drugs adversely influence cardiac electrophysiology by inhibiting repolarising K(+) currents mediated by channels encoded by the human ether-a-go-go-related gene (hERG). In this study, pharmacological blockade of hERG K(+) channel current (I(hERG)) by a novel investigative serotonin-selective reuptake inhibitor (SSRI), CONA-437, was investigated. Whole-cell patch-clamp measurements of I(hERG) were made from human embryonic kidney (HEK 293) cells expressing wild-type (WT) or mutant forms of the hERG channel. With a step-ramp voltage-command, peak I(hERG) was inhibited with an IC(50) of 1.34 µM at 35 ±1°C; the IC(50) with the same protocol was not significantly different at room temperature. Voltage-command waveform selection had only a modest effect on the potency of I(hERG) block: the IC50 with a ventricular action potential command was 0.72 µM. I(hERG) blockade developed rapidly with time following membrane depolarisation and showed a weak dependence on voltage, accompanied by a shift of ≈ -5 mV in voltage-dependence of activation. There was no significant effect of CONA-437 on voltage-dependence of I(hERG) inactivation, though at some voltages an apparent acceleration of the time-course of inactivation was observed. Significantly, mutation of the S6 aromatic amino acid residues Y652 and F656 had only a modest effect on I(hERG) blockade by CONA-437 (a 3-4 fold shift in affinity). CONA-437 at up to 30 µM had no significant effect on either Nav1.5 sodium channels or L-type calcium channels. In conclusion, the novel SSRI CONA-437 is particularly notable as a gating-dependent hERG channel inhibitor for which neither S6 aromatic amino-acid constituent of the canonical drug binding site on the hERG channel appears obligatory for I(hERG) inhibition to occur.

Inhibition of the HERG potassium channel by the tricyclic antidepressant doxepin.

HERG (human ether-à-go-go-related gene) encodes channels responsible for the cardiac rapid delayed rectifier potassium current, I(Kr). This study investigated the effects on HERG channels of doxepin, a tricyclic antidepressant linked to QT interval prolongation and cardiac arrhythmia. Whole-cell patch-clamp recordings were made at 37 degrees C of recombinant HERG channel current (I(HERG)), and of native I(Kr) 'tails' from rabbit ventricular myocytes. Doxepin inhibited I(HERG) with an IC(50) value of 6.5+/-1.4 microM and native I(Kr) with an IC(50) of 4.4+/-0.6 microM. The inhibitory effect on I(HERG) developed rapidly upon membrane depolarization, but with no significant dependence on voltage and with little alteration to the voltage-dependent kinetics of I(HERG). Neither the S631A nor N588K inactivation-attenuating mutations (of residues located in the channel pore and external S5-Pore linker, respectively) significantly reduced the potency of inhibition. The S6 point mutation Y652A increased the IC(50) for I(HERG) blockade by approximately 4.2-fold; the F656A mutant also attenuated doxepin's action at some concentrations. HERG channel blockade is likely to underpin reported cases of QT interval prolongation with doxepin. Notably, this study also establishes doxepin as an effective inhibitor of mutant (N588K) HERG channels responsible for variant 1 of the short QT syndrome.

Regulation of a G protein-gated inwardly rectifying K+ channel by a Ca(2+)-independent protein kinase C.

1. Members of the Kir3.0 family of inwardly rectifying K(+) channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by G(betagamma) subunits released in response to G(i/o)-coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the G(q/11)-coupled M(1) and M(3) muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal-type channels stably expressed in the human embryonic kidney cell line HEK293. 2. We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M(1) and M(3) receptors. This contrasts with the simple stimulation seen through activation of M(2) and M(4) receptors. 3. Channel stimulation via M(1) but not M(3) receptors was sensitive to pertussis toxin whereas channel inhibition through both M(1) and M(3) receptors was insensitive. In contrast over-expression of the C-terminus of phospholipase Cbeta1 or a G(q/11)-specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase. 4. The inhibitory effects of M(1) and M(3) receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4alphaphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M(3) receptor. 5. The receptor-mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0-669 nM). 6. The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCdelta and PKCepsilon. 7. We also demonstrate that activation of such a pathway via both receptor-mediated and receptor-independent means profoundly attenuated subsequent channel stimulation by G(i/o)-coupled receptors. 8. Our data support a role for a Ca(2+)-independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M(1) and M(3) muscarinic receptor stimulation.

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel.

Inwardly rectifying potassium (K(+)) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by G(betagamma) subunits released from G protein heterotrimers of the G(i/o) family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein alpha subunits (G(ialpha1-3) and G(oalphaA)) in mediating coupling between various receptor systems (A(1), alpha(2A), D(2S), M(4), GABA(B)1a+2, and GABA(B)1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant G(i/oalpha) subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A(1), alpha(2A)) known to interact with all isoforms, G(ialpha1-3) and G(oalphaA) can all support a significant degree of coupling to Kir3.1+3.2A. The M(4) receptor appears to preferentially couple to G(ialpha2) while another group of receptors (D(2S), GABA(B)1a+2, GABA(B)1b+2) activates the channel predominantly through G(betagamma) liberated from G(oA) heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABA(B)1. Our data reveal selective pathways of receptor activation through different G(i/oalpha) isoforms for stimulation of the G protein-gated inwardly rectifying K(+) channel.

The G protein alpha subunit has a key role in determining the specificity of coupling to, but not the activation of, G protein-gated inwardly rectifying K(+) channels.

In neuronal and atrial tissue, G protein-gated inwardly rectifying K(+) channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate. They are activated by Gbetagamma dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the G(i/o) family but not receptors coupled to the stimulatory G protein G(s). We have used biochemical, electrophysiological, and molecular biology techniques to examine this specificity of channel activation. In this study we have succeeded in reconstituting such specificity in an heterologous expression system stably expressing a cloned counterpart of the neuronal channel (Kir3.1 and Kir3.2A heteromultimers). The use of pertussis toxin-resistant G protein alpha subunits and chimeras between G(i1) and G(s) indicate a central role for the G protein alpha subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K(+) channels.

The molecular assembly of ATP-sensitive potassium channels. Determinants on the pore forming subunit.

ATP-sensitive potassium channels form a link between membrane excitability and cellular metabolism. These channels are important in physiological processes such as insulin release and they are an important site of drug action. They are an octomeric complex comprised of four sulfonylurea receptors, a member of the ATP-binding cassette family of proteins, and four Kir 6.0 subunits from the inward rectifier family of potassium channels. We have investigated the nature of the interaction between SUR1 and Kir 6.2 and the domains on the channel responsible for the biochemical and functional manifestations of coupling. The results point to the proximal C terminus determining biochemical interaction in a region that also largely governs homotypic and heterotypic interaction between different Kir family members. While this domain may be necessary for functional communication between the two proteins, it is not sufficient since relative modifications of either the N or C terminus are able to disrupt many aspects of functional coupling mediated by the sulfonylurea receptor.

A swelling-activated chloride current in rat sympathetic neurones.

1. We have tested whether neurones show a swelling-induced Cl- current following hypotonic shock, by recording membrane current responses and cell volume changes in voltage clamped isolated rat sympathetic neurones during application of hypotonic solutions. 2. Using both whole-cell and perforated patch recording methods, hypotonic solution caused cell swelling and the activation of an inward Cl- current at -60 mV. This current showed weak outward rectification with no obvious time dependence. It was inhibited by SITS (0.3-1 mM), NPPB (30-300 microM) and niflumic acid (50-200 microM), but not by tamoxifen (10 microM). 3. Hypotonic solution did not cause a rise in intracellular Ca2+ concentration as measured by simultaneous indo-1 fluorescence. Also, neither the volume change nor Cl- current were affected by the removal of external Ca2+ or internal Ca2+ buffering to < or = 1 nM with EGTA. 4. The Cl- current was unaffected by an inhibitor of protein kinase C (PKC; GF109203X, 3 microM) or by omission of ATP from the pipette solution. 5. Cells exhibited a regulatory volume decrease during sustained exposure to hypotonic solution. This was completely inhibited by 0.5 mM niflumic acid. 6. It is concluded that osmotic swelling induces an outwardly rectifying, Ca2(+)- and PKC-independent Cl- current in these nerve cells. It is suggested that this current may be involved in volume regulatory mechanisms.

Synergistic regulation of a neuronal chloride current by intracellular calcium and muscarinic receptor activation: a role for protein kinase C.

Using perforated patch recordings in combination with intracellular Ca2+ ([Ca2+]i) fluorescence measurements, we have identified a delayed Ca(2+)-dependent Cl- current in a mammalian sympathetic ganglion cell. This Cl- current is induced by the synergistic action of Ca2+ and diacylglycerol (DAG) and is blocked by inhibitors of protein kinase C. As a result, the current can be induced by acetylcholine through the conjoint activation of nicotinic receptors (to produce a rise in [Ca2+]i) and muscarinic receptors (to generate DAG). This demonstrates an unusual form of synergism between the two effects of a single transmitter mediated via separate receptors operating within a time scale that could be of physiological significance.